Name: multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos
Uploaded: 2017-08-09T15:00:00
Description: Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Forfatterne takker Thomas Schilling for vennlig gifting vs (sox10:eGFP) fisk og Mary Halloran for vennlige gifting vs(foxd3:GFP) fisken.

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
<ol>
	<li>Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. 
	NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G...

<ol>
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Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.</a> Prenat Diagn. 33 (1), 75-80 (2013).</li><li>Aliferis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+nonsense+B3GALTL+mutation+confirms+Peters+plus+syndrome+in+a+patient+with+multiple+malformations+and+Peters+anomaly.">A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.</a> Ophthalmic Genet. 31 (4), 205-208 (2010).</li><li>Lesnik Oberstein, ., S, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peters+Plus+syndrome+is+caused+by+mutations+in+B3GALTL,+a+putative+glycosyltransferase.">Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase.</a> Am J Hum Genet. 79 (3), 562-566 (2006).</li><li>Brittijn, S. 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Flere Foton tidsinnstilt bildebehandling kan i vivo sporing av forbigående og vandrende celle populasjoner. Denne kraftige teknikken kan brukes til å studere embryonale prosesser i sanntid, og studien, resultatene av denne metoden forbedret gjeldende kunnskap om neural crest celle migrasjon og utvikling. Tidligere time-lapse imaging studier vanligvis benytte AC confocal mikroskopi for laserskanning. 29 , 30 , 31 ...

Medfødt øyesykdommer kan forårsake barndommen blindhet og er ofte på grunn av unormalt av skallen neural kam. Neural crest celler er forbigående stamceller som oppstår fra medfødte og danne flere vev i kroppen. 1 , 2 , 3 , 4 , 5 Neural crest celler, avledet fra prosencephalon og mesencephalon, gi opphav til beina og brusken til midface og frontal regioner, iris, hornhinnen, trabekulært meshwork, og sclera i anterior segment av øyet. 4 , 6 , 7 , 8 Neural crest celler fra rhombencephalon form disse strukturene buer, kjeven og kardiale utløp skrift. 1 , 3 , 4 , 9 , 10 studier har fremhevet bidrag av neural kam å okulær og periocular utvikling, understreker viktigheten av disse cellene i virveldyr øye utvikling. Faktisk avbrudd av neural crest celle migrasjon og differensiering føre til craniofacial og okulær avvik observert i Axenfeld-Rieger syndrom og Peters pluss syndrom. 11 , 12 , 13 , 14 , 15 , 16 , 17 derfor, en omfattende forståelse av migrasjon, spredning og differensiering av disse neural crest celler vil gi innsikt i kompleksiteten underliggende medfødt øyesykdommer.
Sebrafisk er en effektiv modell organisme for å studere okulær utvikling, strukturer av sebrafisk øyet er lik for pattedyr i papirformat, og mange gener er evolutionarily konservert mellom sebrafisk og pattedyr. 18 , 19 , 20 i tillegg sebrafisk embryoer er gjennomsiktig og oviparous, tilrettelegge visualisering av øye utvikling i sanntid.
Utvide på tidligere publiserte arbeid,6,7,20 vandrende mønster av neural crest celler ble beskrevet ved hjelp av flere Foton fluorescens time-lapse imaging transgene sebrafisk linjene merket med grønne fluorescerende protein (GFP) under transcriptional kontroll av SRY (sex-bestemme region Y)-boks 10 (sox10) eller Forkhead for D3 (foxd3) genet regulatoriske regioner. 21 , 22 , 23 , 24. flere Foton fluorescens tidsinnstilt bildebehandling er en kraftfull teknikk som kombinerer avansert optisk teknikker av laserskanning mikroskopi med lang bølgelengde multi Foton fluorescens eksitasjon å ta høy oppløsning, tredimensjonale bilder av merket med fluorophores. 25 , 26 , 27 bruk av flere Foton laseren har klare fordeler fremfor standard AC confocal mikroskopi, inkludert økt vev gjennomtrengning og nedsatt fluorophore bleking.
Bruker denne metoden, ble to forskjellige populasjoner av neural crest celler i timingen av migrasjon og vandrende veier diskriminert, nemlig foxd3-positive neural crest cellene i periocular mesenchyme og utvikle øye og sox10-positive neural crest i den craniofacial mesenchyme. Med denne metoden er en tilnærming til å visualisere overføringen for okulær og craniofacial neural crest sebrafisk introdusert, gjør det enkelt å observere regulert neural crest overføring i sanntid under utvikling.
Denne protokollen gir informasjon for å generere time-lapse videoer under øyet utviklingen i vs (sox10:EGFP) og vs (foxd3:GFP) transgene sebrafisk, som et eksempel. Denne protokollen kan videre brukes for høy oppløsning, tredimensjonale, sanntids visualisering av utviklingen av alle okulær og craniofacial struktur avledet fra neural crest celler i sebrafisk. Denne metoden kan videre ytterligere brukes for visualisering av utviklingen av andre vev og organer i sebrafisk og andre dyr modeller.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
			<td style="width:175px;">Aquaneering</td>
			<td style="width:104px;">ZHCT100</td>
			<td style="width:391px;">Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">M205 FA Combi-Scope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;">Stereofluorescence Microscope - FusionOptics and TripleBeam</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">7760-5KG</td>
			<td style="width:391px;">Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">1049380500</td>
			<td style="width:391px;">Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">C79-500</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 10035-04-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Magnesium Sulfate (Anhydrous)</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">MX0075-1</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methylene Blue</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">284-12</td>
			<td style="width:391px;">Glass bottle; 25 g. Powder, Certified Biological Stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">SX0320-1</td>
			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N-Phenylthiourea</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">P7629-25G</td>
			<td style="width:391px;">&#62;98%. CAS Number 103-85-5, EC Number 203-151-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">D8418-500ML</td>
			<td style="width:391px;">Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
			<td style="width:175px;">Western Chemical Inc.</td>
			<td style="width:104px;">MS222</td>
			<td style="width:391px;">Tricaine-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Low-Melt Agarose</td>
			<td style="width:175px;">ISC Bioexpress</td>
			<td style="width:104px;">E-3112-25</td>
			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Open Bath Chamber</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">RC-40HP</td>
			<td style="width:391px;">High Profile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass Coverslips</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">12-545-102</td>
			<td style="width:391px;">Circle cover glass. 25 mm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">High Vacuum Grease</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">14-635-5C</td>
			<td style="width:391px;">2.0-lb. tube. DOW CORNING CORPORATION 
			1658832</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Quick Exchange Platform</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">QE-1</td>
			<td style="width:391px;">35 mm</td>
		</tr>
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			<td height="21" style="height:21px;width:251px;">Stage Adapter</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">SA-20LZ-AL</td>
			<td style="width:391px;">16.5 x 10 cm</td>
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			<td height="40" style="height:40px;width:251px;">TC SP5 MP multi-photon microscope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mai Tai DeepSee Ti-Sapphire Laser</td>
			<td style="width:175px;">SpectraPhysics</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Laser Safety Box</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Leica Application Suite X (LAS X)&#160; Software</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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			<td height="42" style="height:42px;width:251px;">Photoshop CS 6 Version 13.0 x64 Software</td>
			<td style="width:175px;">Adobe</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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			<td height="21" style="height:21px;width:251px;">iMovie Version 10.1.4 Software</td>
			<td style="width:175px;">Apple</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Medfødt øye og craniofacial anomalier gjenspeiler forstyrrelser i neural toppen, en forbigående befolkningen i vandrende stamceller som gir opphav til mange celletyper i kroppen. Forstå biologi av neural kam er begrenset, gjenspeiler en mangel på genetisk medgjørlig modeller som kan være studert i vivo og i sanntid. Sebrafisk er en særlig viktig utviklingsmessige modellen for å studere vandrende celle populasjoner, som neural toppen. For å undersøke neural crest overføringen i det tredje øyet, ble en kombinasjon av avansert optisk teknikker av laserskanning mikroskopi med lang bølgelengde multi Foton fluorescens eksitasjon implementert for å ta med høy oppløsning, tredimensjonale, sanntid videoer av tredje øyet i transgene sebrafisk embryoer, nemlig vs (sox10:EGFP) og vs (foxd3:GFP), som sox10 og foxd3 har vist mange dyr modeller å regulere tidlig neural crest differensiering og trolig representere markører for neural crest celler. Flere Foton tidsinnstilt bildebehandling ble brukt til å skjelne atferd og vandrende mønstre av to neural crest celle populasjoner bidra til tidlig øye utvikling. Denne protokollen gir informasjon for å generere time-lapse videoer under sebrafisk neural crest overføring, som et eksempel, og kan brukes til ytterligere for å visualisere den tidlige utviklingen av mange strukturer i sebrafisk og andre modellen organismer.

Flere Foton fluorescens tidsinnstilt bildebehandling generert en rekke videoer som avslørte migrasjon av skallen neural crest celler som gir opphav til craniofacial strukturer og anterior segment av øyet i vs (sox10:EGFP) og vs (foxd3:GFP) sebrafisk linjer. Som et eksempel overføre sox10 -positive neural crest celler mellom 12 og 30 hpf fra kanten av medfødte i den craniofacial regionen (Video 1, figur 2). <str...

Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Flere Foton tid forfalle Imaging se utvikling i sanntid: visualisering av migrere Neural Crest celler i sebrafisk embryo

En kombinasjon av avansert optisk teknikker av laserskanning mikroskopi med lang bølgelengde multi Foton fluorescens eksitasjon ble gjennomført for å fange høy oppløsning, tredimensjonale, sanntid bildebehandling for neural crest vs (sox10:EGFP) og vs (foxd3:GFP) sebrafisk embryoer.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyserer<em&gt; I Vivo</em&gt; Cell Migration bruker Cell transplantasjoner og Time-lapse Imaging in Sebrafisk embryo

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Analyse av Sebrafisk Nyre Development med Time-lapse Imaging Ved hjelp av en dissekere mikroskop Utstyrt for Optical Seksjonering

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Real Time Kinetic Måling og visualisering av somatiske Omprogrammering

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Induksjon av hypoksi i levende frosk og sebrafiskembryoer

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Utvikling av In Vitro analysen å Quantitate blodkreft stilk og stamfar celler (HSPCs) i utvikle sebrafisk embryo

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Sanntid Live-celle flowcytometri å undersøke kalsium tilstrømningen Pore dannelse og fagocytose av P2X7 reseptorer i voksen nevrale stamceller

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

En lagdelt montering metode for utvidet tidsforløp Konfokalmikroskopi mikroskopi av hele sebrafisk embryo

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Real-Time bioluminesens Imaging av hakk signalering dynamikk under murine neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

Isolasjon, kultur og adipogen induksjon av Neural Crest Original Adipose-Avledet stamceller fra periaortisk fettvev

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...